Bottom Line:
Using ratiometric calcium imaging, we found that ADP evokes increases in intracellular calcium in isolated DRG neurons and also produces a pertussis toxin-sensitive inhibition of depolarization-evoked calcium transients.The inhibitory effect of ADP was unaltered in the presence of the selective P2Y1 antagonist MRS2179 and in neurons isolated from P2Y1 knockout mice, whereas ADP-evoked calcium transients were greatly reduced.Taken together, our data suggest that Gi-coupled P2Y receptors are broadly expressed in nociceptors, inhibit nociceptive signaling in vivo, and represent potential targets for the development of novel analgesic drugs.

Background: Investigations of nucleotide signaling in nociception to date have focused on actions of adenosine triphosphate (ATP). Both ATP-gated ion channels (P2X receptors) and G protein-coupled (P2Y) receptors contribute to nociceptive signaling in peripheral sensory neurons. In addition, several studies have implicated the Gq-coupled adenosine diphosphate (ADP) receptor P2Y1 in sensory transduction. In this study, we examined the expression and function of P2Y1 and the Gi-coupled receptors P2Y12, P2Y13 and P2Y14 in sensory neurons to determine their contribution to nociception.

Results: We detected mRNA and protein for ADP receptors P2Y12 and P2Y13 in mouse dorsal root ganglia (DRG). P2Y14, a homologous Gi-coupled nucleotide receptor, is also expressed in DRG. Immunohistochemical analysis of receptor distribution indicated that these receptors are widely expressed in nociceptive neurons. Using ratiometric calcium imaging, we found that ADP evokes increases in intracellular calcium in isolated DRG neurons and also produces a pertussis toxin-sensitive inhibition of depolarization-evoked calcium transients. The inhibitory effect of ADP was unaltered in the presence of the selective P2Y1 antagonist MRS2179 and in neurons isolated from P2Y1 knockout mice, whereas ADP-evoked calcium transients were greatly reduced. Analysis of behavioral responses to noxious heat before and after inflammatory injury (injection of complete Freund's adjuvant into the hindpaw) revealed that P2Y1 is required for the full expression of inflammatory hyperalgesia, whereas local injection of agonists for Gi-coupled P2Y receptors reduced hyperalgesia.

Conclusions: We report that Gi-coupled P2Y receptors are widely expressed in peripheral sensory neurons. Agonists for these receptors inhibit nociceptive signaling in isolated neurons and reduce behavioral hyperalgesia in vivo. Anti-nociceptive actions of these receptors appear to be antagonized by the Gq-coupled ADP receptor, P2Y1, which is required for the full expression of inflammatory hyperalgesia. We propose that nociceptor sensitivity is modulated by the integration of nucleotide signaling through Gq- and Gi-coupled P2Y receptors, and this balance is altered in response to inflammatory injury. Taken together, our data suggest that Gi-coupled P2Y receptors are broadly expressed in nociceptors, inhibit nociceptive signaling in vivo, and represent potential targets for the development of novel analgesic drugs.

Figure 4: The inhibitory effect of ADP is enhanced in the absence of P2Y1 signaling. The magnitudes of depolarization-evoked Ca++ transients in sensory neurons were measured before, and 5, 10 and 15 minutes after agonist application. Ca++ transients were not affected by application of buffer alone (dashed lines), but were significantly reduced after application of ADP (A-B), IDP (C), or UDPG (D; solid lines). Inhibition was prolonged by application of the selective P2Y1 antagonist MRS2179 (A) and in neurons from P2Y1-/- mice (B). There was no effect of MRS2179 in P2Y1-/- neurons (B). Values are mean ± SEM; n = 10 mice/timepoint each treatment. *p < 0.02 versus control for each treatment.

Mentions:
Neurons inhibited by nucleotides were tested for responsiveness to 1 μM capsaicin to test for expression of the capsaicin receptor TRPV1 as a marker for a subset of nociceptors [17]. A large proportion of capsaicin-responsive neurons was inhibited by nucleotides, consistent with our hypothesis that P2YGi receptors have inhibitory actions in nociceptive sensory neurons (Table 3). UDPG inhibited the largest proportion of capsaicin-responsive neurons, followed by ADP and IDP. Application of ADP, IDP or UDPG resulted in long-lasting inhibition in many cells. Responses at 5, 10 and 15 minutes post-agonist application were compared to peak responses to depolarizing stimuli obtained prior to P2YGi receptor activation; these data are shown in Figure 4. This reduction in response magnitude does not reflect run-down, as cells not treated with nucleotides showed no diminution of depolarizing responses. These data suggest that P2YGi activation may have analgesic effects in vivo.

Figure 4: The inhibitory effect of ADP is enhanced in the absence of P2Y1 signaling. The magnitudes of depolarization-evoked Ca++ transients in sensory neurons were measured before, and 5, 10 and 15 minutes after agonist application. Ca++ transients were not affected by application of buffer alone (dashed lines), but were significantly reduced after application of ADP (A-B), IDP (C), or UDPG (D; solid lines). Inhibition was prolonged by application of the selective P2Y1 antagonist MRS2179 (A) and in neurons from P2Y1-/- mice (B). There was no effect of MRS2179 in P2Y1-/- neurons (B). Values are mean ± SEM; n = 10 mice/timepoint each treatment. *p < 0.02 versus control for each treatment.

Mentions:
Neurons inhibited by nucleotides were tested for responsiveness to 1 μM capsaicin to test for expression of the capsaicin receptor TRPV1 as a marker for a subset of nociceptors [17]. A large proportion of capsaicin-responsive neurons was inhibited by nucleotides, consistent with our hypothesis that P2YGi receptors have inhibitory actions in nociceptive sensory neurons (Table 3). UDPG inhibited the largest proportion of capsaicin-responsive neurons, followed by ADP and IDP. Application of ADP, IDP or UDPG resulted in long-lasting inhibition in many cells. Responses at 5, 10 and 15 minutes post-agonist application were compared to peak responses to depolarizing stimuli obtained prior to P2YGi receptor activation; these data are shown in Figure 4. This reduction in response magnitude does not reflect run-down, as cells not treated with nucleotides showed no diminution of depolarizing responses. These data suggest that P2YGi activation may have analgesic effects in vivo.

Bottom Line:
Using ratiometric calcium imaging, we found that ADP evokes increases in intracellular calcium in isolated DRG neurons and also produces a pertussis toxin-sensitive inhibition of depolarization-evoked calcium transients.The inhibitory effect of ADP was unaltered in the presence of the selective P2Y1 antagonist MRS2179 and in neurons isolated from P2Y1 knockout mice, whereas ADP-evoked calcium transients were greatly reduced.Taken together, our data suggest that Gi-coupled P2Y receptors are broadly expressed in nociceptors, inhibit nociceptive signaling in vivo, and represent potential targets for the development of novel analgesic drugs.

Background: Investigations of nucleotide signaling in nociception to date have focused on actions of adenosine triphosphate (ATP). Both ATP-gated ion channels (P2X receptors) and G protein-coupled (P2Y) receptors contribute to nociceptive signaling in peripheral sensory neurons. In addition, several studies have implicated the Gq-coupled adenosine diphosphate (ADP) receptor P2Y1 in sensory transduction. In this study, we examined the expression and function of P2Y1 and the Gi-coupled receptors P2Y12, P2Y13 and P2Y14 in sensory neurons to determine their contribution to nociception.

Results: We detected mRNA and protein for ADP receptors P2Y12 and P2Y13 in mouse dorsal root ganglia (DRG). P2Y14, a homologous Gi-coupled nucleotide receptor, is also expressed in DRG. Immunohistochemical analysis of receptor distribution indicated that these receptors are widely expressed in nociceptive neurons. Using ratiometric calcium imaging, we found that ADP evokes increases in intracellular calcium in isolated DRG neurons and also produces a pertussis toxin-sensitive inhibition of depolarization-evoked calcium transients. The inhibitory effect of ADP was unaltered in the presence of the selective P2Y1 antagonist MRS2179 and in neurons isolated from P2Y1 knockout mice, whereas ADP-evoked calcium transients were greatly reduced. Analysis of behavioral responses to noxious heat before and after inflammatory injury (injection of complete Freund's adjuvant into the hindpaw) revealed that P2Y1 is required for the full expression of inflammatory hyperalgesia, whereas local injection of agonists for Gi-coupled P2Y receptors reduced hyperalgesia.

Conclusions: We report that Gi-coupled P2Y receptors are widely expressed in peripheral sensory neurons. Agonists for these receptors inhibit nociceptive signaling in isolated neurons and reduce behavioral hyperalgesia in vivo. Anti-nociceptive actions of these receptors appear to be antagonized by the Gq-coupled ADP receptor, P2Y1, which is required for the full expression of inflammatory hyperalgesia. We propose that nociceptor sensitivity is modulated by the integration of nucleotide signaling through Gq- and Gi-coupled P2Y receptors, and this balance is altered in response to inflammatory injury. Taken together, our data suggest that Gi-coupled P2Y receptors are broadly expressed in nociceptors, inhibit nociceptive signaling in vivo, and represent potential targets for the development of novel analgesic drugs.